Paired macromolecule abundance and t-cell receptor sequencing with high spatial resolution

Abstract

The present disclosure relates to compositions and methods for assessing extended length T-cell receptor (TCR) transcript sequences (i.e., TCR transcript sequences that span TCR transcript variable regions) in a spatially-defined manner across a tissue sample, specifically providing for obtaining useful TCR sequences at high spatial resolution while also assessing relative macromolecule abundance (e.g., RNA expression levels) with deep transcriptomic coverage at similarly high-resolution across the tissue sample.

We claim: 
     
         1 . A method for obtaining from a tissue sample spatially-resolvable T cell receptor (TCR) sequence that spans TCR transcript variable regions, the method comprising:
 (i) obtaining a tissue sample from a subject;   (ii) preparing a section of the tissue sample;   (iii) providing a solid support;   (iv) contacting the solid support with a capture material, thereby forming a capture material-coated solid support;   (v) contacting the capture material-coated solid support with a population of 1-100 μm diameter beads, wherein each bead has at least 1000 attached oligonucleotides and wherein at least one attached oligonucleotide of each bead each comprises: (a) a bead identification sequence that is common to all at least 1000 oligonucleotides on each bead and (b) a poly-dT tail of sufficient length to allow for capture of poly-A-tailed RNAs via hybridization, wherein the bead identification sequence that is common to all at least 1000 oligonucleotides on each bead is either a bead identification sequence that is unique to each bead within the population of 1-100 μm diameter beads or is a bead identification sequence that is a member of a population of bead identification sequences that is sufficiently degenerate to the population of 1-100 μm diameter beads that a majority of beads within the population of 1-100 μm diameter beads each possesses a unique bead identification sequence, thereby capturing a subpopulation of the population of 1-100 μm diameter beads upon the solid support;   (vi) identifying the bead identification sequence and associated two-dimensional position on the solid support of individual beads of the subpopulation of beads attached to the solid support;   (vii) contacting the subpopulation of 1-100 μm diameter beads captured upon the solid support with of the tissue sample;   (viii) performing a reverse transcription reaction upon poly-A-tailed RNAs captured by the bead subpopulation, thereby generating a cDNA population;   (ix) contacting a selection of or all of the cDNA population with (a) RNase H-dependent PCR primers designed for specific amplification of TCR-alpha and TCR-beta cDNAs and (b) RNase H, and performing PCR amplification upon the cDNA population, thereby generating a PCR-amplified nucleic acid population enriched for TCR-alpha and TCR-beta sequences; and   (x) obtaining sequence from the PCR-amplified nucleic acid population enriched for TCR-alpha and TCR-beta sequences using a sequencing process for TCR sequence-containing PCR-amplified nucleic acids having an average read length on at least one end in excess of 200 nucleotides, thereby obtaining TCR sequences that span TCR transcript variable regions for substantially all TCR sequences obtained and obtaining sequence from the PCR-amplified nucleic acid population enriched for TCR-alpha and TCR-beta sequences of bead identification sequences associated with TCR sequences,   thereby obtaining from the tissue sample spatially-resolvable T cell receptor (TCR) sequence that spans TCR transcript variable regions.   
     
     
         2 . The method of  claim 1 , wherein each bead has at least 1000 attached oligonucleotides and wherein at least 100, optionally at least 1000, attached oligonucleotides of each bead each comprises: (a) a bead identification sequence that is common to all at least 1000 oligonucleotides on each bead and (b) a poly-dT tail of sufficient length to allow for capture of poly-A-tailed RNAs via hybridization. 
     
     
         3 . The method of  claim 2 , wherein PCR amplification is performed upon the cDNA population of step (viii) in a manner that does not specifically enrich for TCR-alpha and TCR-beta sequences, thereby generating a PCR-amplified cDNA population that is not specifically enriched for TCR-alpha and TCR-beta sequences, wherein the PCR-amplified cDNA population that is not specifically enriched for TCR-alpha and TCR-beta sequences, or a subpopulation thereof, is the cDNA population contacted in step (ix) with (a) RNase H-dependent PCR primers designed for specific amplification of TCR-alpha and TCR-beta cDNAs and (b) RNase H, thereby generating a PCR-amplified nucleic acid population enriched for TCR-alpha and TCR-beta sequences. 
     
     
         4 . The method of  claim 2 , wherein the cDNA population of step (viii) is partitioned into a first selection of the cDNA population that is contacted in step (ix) with RNase H-dependent PCR primers designed for specific amplification of TCR-alpha and TCR-beta cDNAs and RNase H, thereby generating a first PCR-amplified nucleic acid population that is enriched for TCR-alpha and TCR-beta sequences, and a second selection of the cDNA population, optionally wherein the second selection of the cDNA population is amplified with primers that are not selective for TCR sequence, thereby generating a second PCR-amplified nucleic acid population that is not enriched for TCR sequence relative to the cDNA population of step (viii). 
     
     
         5 . The method of  claim 3 , wherein the PCR-amplified nucleic acid population enriched for TCR-alpha and TCR-beta sequences and the PCR-amplified nucleic acid population that is not specifically enriched for TCR-alpha and TCR-beta sequences are combined prior to obtaining sequence from the PCR-amplified nucleic acid population using a sequencing process having an average read length in excess of 200 nucleotides in step (x) for at least a TCR sequence-containing end of a TCR sequence-containing PCR-amplified nucleic acid, wherein sequences of non-TCR transcripts and associated bead identification sequences are thereby also obtained in step (x), wherein the method thereby obtains both spatially-resolvable T cell receptor (TCR) sequence that spans TCR transcript variable regions and spatially-resolvable transcript abundance information from the tissue sample. 
     
     
         6 . The method of  claim 1 , wherein the PCR-amplified nucleic acid population enriched for TCR-alpha and TCR-beta sequences, and optionally the PCR-amplified nucleic acid population that is not specifically enriched for TCR-alpha and TCR-beta sequences, is cleaved and tagged prior to obtaining sequence from the PCR-amplified nucleic acid population in step (x). 
     
     
         7 . The method of  claim 1 , wherein bead identification sequences associated with transcripts are obtained using paired-end sequencing, optionally wherein sequences of bead identification sequences associated with TCR sequences are obtained using paired-end sequencing. 
     
     
         8 . The method of  claim 1 , wherein a subpopulation of the at least 1000 attached oligonucleotides of each bead comprises (a) a bead identification sequence that is common to all at least 1000 oligonucleotides on each bead and (b) a macromolecule-specific capture sequence that does not comprise a poly-dT tail. 
     
     
         9 . The method of  claim 8 , wherein the macromolecule is selected from the group consisting of RNA, DNA and protein. 
     
     
         10 . The method of  claim 8 , wherein the macromolecule-specific capture sequence comprises a gene-specific or transcript-specific sequence. 
     
     
         11 . The method of  claim 9 , wherein the DNA is selected from the group consisting of a genomic DNA and a barcode DNA. 
     
     
         12 . The method of  claim 8 , wherein the macromolecule-specific capture sequence is a component of a loaded transposase. 
     
     
         13 . The method of  claim 8 , wherein a DNA barcode is used to capture an attached protein, optionally wherein the barcode-attached protein is an antibody, optionally wherein the antibody is specifically bound to a target protein, optionally wherein the antibody-bound target protein comprises a label. 
     
     
         14 . The method of  claim 8 , further comprising PCR amplifying a nucleotide sequence of the captured macromolecule, thereby generating a PCR-amplified macromolecule nucleotide sequence population, and obtaining sequence from the PCR-amplified macromolecule nucleotide sequence population, thereby also obtaining spatially-resolvable macromolecule abundance data from the tissue sample. 
     
     
         15 . The method of  claim 1 , wherein the PCR-amplified nucleic acid population comprising TCR-alpha and TCR-beta sequences is cleaved and tagged before obtaining sequence from the PCR-amplified nucleic acid population in step (x), optionally wherein a second PCR-amplified nucleic acid population is also cleaved and tagged before also obtaining sequence from the second PCR-amplified nucleic acid population. 
     
     
         16 . The method of  claim 1 , wherein the obtaining sequence from the PCR-amplified nucleic acid population in step (x) is performed using a next-generation sequencing (NGS) method, optionally wherein the NGS sequencing method is selected from the group consisting of solid-phase, reversible dye-terminator sequencing; massively parallel signature sequencing; pyro-sequencing; sequencing-by-ligation; ion semiconductor sequencing; Nanopore sequencing and DNA nanoball sequencing, optionally wherein the next-generation sequencing approach is solid-phase, reversible dye-terminator sequencing. 
     
     
         17 . The method of  claim 1 , wherein the obtaining sequence from the PCR-amplified nucleic acid population in step (x) is performed using a long read sequencing (LRS) method, optionally wherein the LRS method is selected from the group consisting of single molecule real time sequencing (SMRT) and nanopore sequencing. 
     
     
         18 . The method of  claim 1 , wherein:
 the average read length of the sequencing process exceeds about 850 nucleotides, optionally wherein the average read length of the sequencing process exceeds about 900 nucleotides, optionally wherein the average read length of the sequencing process exceeds about 950 nucleotides, optionally wherein the average read length of the sequencing process exceeds about 1000 nucleotides, optionally wherein the average read length of the sequencing process exceeds about 1050 nucleotides, optionally wherein the average read length of the sequencing process exceeds about 1100 nucleotides, optionally wherein the average read length of the sequencing process exceeds about 1150 nucleotides, optionally wherein the average read length of the sequencing process exceeds about 1200 nucleotides, optionally wherein the average read length of the sequencing process exceeds about 1250 nucleotides, optionally wherein the average read length of the sequencing process exceeds about 1300 nucleotides;   the tissue sample is obtained from a tissue selected from the group consisting of brain, lung, liver, kidney, pancreas, heart, spleen, lymph node, thymus and tumor;   the subject is a mammal, optionally a human;   the tissue sample is fixed, optionally wherein the tissue sample is fixed with a fixative selected from the group consisting of formalin, methanol, ethanol and acetone, optionally the tissue sample is a formalin-fixated and paraffin-embedded (FFPE) pathology specimen;   the solid support is a slide, optionally the solid support is a glass slide;   the capture material is applied as a liquid, optionally wherein the capture material is applied using a brush or aerosol spray, optionally wherein the capture material is a liquid electrical tape, optionally wherein the capture material dries to form a vinyl polymer, optionally wherein the vinyl polymer is polyvinyl hexane;   the 1-100 μm diameter beads comprise porous polystyrene, porous polymethacrylate and/or polyacrylamide;   the beads are 1-40 μm diameter beads, optionally wherein the beads are 10 μm beads;   the step of (vi) identifying the bead identification sequence and associated two-dimensional position on the solid support of individual beads of the subpopulation of beads attached to the solid support comprises performance of a sequencing-by-ligation technique;   the subpopulation of 1-100 μm diameter beads captured upon the solid support in step (vii) is maintained at a temperature between 4° C. and 30° C., optionally at about 25° C.;   step (vii) further comprises contacting the subpopulation of 1-100 μm diameter beads captured upon the solid support with a wash solution, optionally with a saline solution, optionally with a solution comprising between about 1M and about 3M NaCl, optionally with a saline-sodium citrate buffer comprising between about 1M and about 3M NaCl;   the bead identification sequence and associated two-dimensional position on the solid support of individual beads of the subpopulation of beads attached to the solid support is registered in a computer;   the method further comprises step (xi) generating an image of the tissue sample that depicts the location(s) and relative abundance of one or more captured TCRs or other captured macromolecules within the sample, optionally wherein the image is a two-dimensional image;   the hybridization is performed in 6×SSC buffer, optionally wherein the 6×SSC buffer is supplemented with detergent;   a selection of the beads possess primers against specific transcripts;   the barcoded array is reusable, optionally wherein cDNA is generated and then the second strand (carrying the barcode location) is synthesized, optionally wherein the second strand is capable of release from the array, optionally wherein the cDNA can be cleaved using a restriction enzyme to reveal a poly(A) tail on the array, thereby allowing for the array to be reused;   transcript-specific amplification of one or more transcripts other than TCR transcripts is also performed;   an array (puck) is physically transferred from one surface to another, optionally wherein a gel encasement is formed on top of the array (puck), thereby allowing beads to be picked up off the surface of the array (puck) without altering bead positions relative to each other;   the beads or array comprise or bind oligonucleotide-conjugated antibodies; and/or   the oligonucleotides having a poly-dT tail of sufficient length to allow for capture of poly-A-tailed RNAs via hybridization comprise unique molecular identifiers (UMIs), optionally wherein the UMIs of the hybridization probes are counted via sequencing to assess the levels of hybridization probe-bound macromolecules, optionally wherein the hybridization probe-bound macromolecules are selected from the group consisting of proteins, exons, transcripts, nucleic acid sequences comprising single nucleotide polymorphisms (SNPs) and/or genomic regions.   
     
     
         19 . A method for obtaining from a tissue sample spatially-resolvable TCR sequence that spans TCR transcript variable regions and spatially-resolvable bulk poly-A-tailed RNA expression data, the method comprising:
 (i) obtaining a tissue sample from a subject;   (ii) preparing a section of the tissue sample;   (iii) obtaining a solid support;   (iv) contacting the solid support with a capture material, thereby forming a capture material-coated solid support;   (v) contacting the capture material-coated solid support with a population of 1-100 μm diameter beads, wherein each bead has at least 1000 attached oligonucleotides and wherein at least 1000 attached oligonucleotides of each bead each comprises: (a) a bead identification sequence that is common to all at least 1000 oligonucleotides on each bead and (b) a poly-dT tail of sufficient length to allow for capture of poly-A-tailed RNAs via hybridization wherein the bead identification sequence that is common to all at least 1000 oligonucleotides on each bead is either a bead identification sequence that is unique to each bead within the population of 1-100 μm diameter beads or is a bead identification sequence that is a member of a population of bead identification sequences that is sufficiently degenerate to the population of 1-100 μm diameter beads that a majority of beads within the population of 1-100 μm diameter beads each possesses a unique bead identification sequence, thereby capturing a subpopulation of the population of 1-100 μm diameter beads upon the solid support;   (vi) identifying the bead identification sequence and associated two-dimensional position on the solid support of individual beads of the subpopulation of beads attached to the solid support;   (vii) contacting the subpopulation of 1-100 μm diameter beads captured upon the solid support with the section of the tissue sample;   (viii) performing a reverse transcription reaction upon poly-A-tailed RNAs captured by the bead subpopulation, thereby generating a cDNA population;   (ix) performing PCR amplification upon the cDNA subpopulation in a manner that does not specifically enrich for TCR-alpha and TCR-beta sequences, thereby generating a PCR-amplified nucleic acid population not specifically enriched for TCR-alpha and TCR-beta sequences;   (x) contacting the PCR-amplified nucleic acid population not specifically enriched for TCR-alpha and TCR-beta sequences, or a subpopulation thereof, with (a) RNase H-dependent PCR primers designed for specific amplification of TCR-alpha and TCR-beta cDNAs and (b) RNase H, and performing PCR amplification, thereby generating a PCR-amplified nucleic acid population enriched for TCR-alpha and TCR-beta sequences;   (xi) combining the PCR-amplified nucleic acid population enriched for TCR-alpha and TCR-beta sequences and the PCR-amplified nucleic acid population not specifically enriched for TCR-alpha and TCR-beta sequences into a single PCR-amplified nucleic acid population; and   (xii) obtaining sequence from the PCR-amplified nucleic acid population using a sequencing process for TCR sequence-containing PCR-amplified nucleic acids having an average read length on at least one end in excess of 200 nucleotides, thereby obtaining (a) TCR sequences that span TCR transcript variable regions for substantially all TCR sequences obtained; (b) sequences of bead identification sequences associated with TCR sequences; and (c) sequences of a population of poly-A-tailed RNAs bound to the bead oligonucleotides and associated bead identification sequences for sequenced poly-A-tailed RNAs,   thereby obtaining from the tissue sample spatially-resolvable T cell receptor (TCR) sequence that spans TCR transcript variable regions and spatially-resolvable bulk poly-A-tailed RNA expression data.   
     
     
         20 . A method selected from the group consisting of:
 A method for obtaining from a tissue sample spatially-resolvable TCR sequence that spans TCR transcript variable regions, the method comprising:   (i) generating a well array, wherein each well of the array can hold exactly one bead;   (ii) depositing beads into the wells of the well array, optionally by evaporation in a centrifuge;   (iii) brushing the well array to remove all of the beads not present in wells;   (iv) obtaining a tissue sample from a subject;   (v) preparing a section of the tissue sample;   (vi) depositing the section onto the well array and centrifuging, thereby forcing the section into the wells of the well array;   (vii) adding digestion buffer, thereby lysing the section and causing the RNA of cells of the section to transfer onto the beads in the wells;   (viii) performing a reverse transcription reaction upon the beads in the wells, thereby generating a cDNA population;   (ix) contacting a selection of or all of the cDNA population with (a) RNase H-dependent PCR primers designed for specific amplification of TCR-alpha and TCR-beta cDNAs and (b) RNase H, and performing PCR amplification upon the cDNA population, thereby generating a PCR-amplified nucleic acid population comprising TCR-alpha and TCR-beta sequences; and   (x) obtaining sequence from the PCR-amplified nucleic acid population using a sequencing process for TCR sequence-containing PCR-amplified nucleic acids having an average read length on at least one end in excess of 200 nucleotides, thereby obtaining TCR sequences that span TCR transcript variable regions for substantially all TCR sequences obtained and obtaining sequence from the PCR-amplified nucleic acid population of bead identification sequences associated with TCR sequences,   optionally further comprising removing beads from the wells by sonication or by photocleavage after step (vii), optionally before performing step (viii),   thereby obtaining from the tissue sample spatially-resolvable T cell receptor (TCR) sequence that spans TCR transcript variable regions;   A method for obtaining from a tissue sample spatially-resolvable TCR sequence that spans TCR transcript variable regions, the method comprising:   (i) obtaining a tissue sample from a subject;   (ii) preparing a section of the tissue sample;   (iii) obtaining a solid support;   (iv) adhering clusters of oligonucleotides in an array attached to the solid support, optionally wherein the array comprises barcoded clusters of oligonucleotides on a surface;   (v) identifying oligonucleotide cluster identification sequences and associated two-dimensional positions on the solid support of individual oligonucleotide clusters attached to the solid support, wherein the individual oligonucleotides are designed to capture RNA or DNA from the section of the tissue sample, optionally wherein at least one of the individual oligonucleotides of each cluster is designed for specific capture of TCR mRNA from the section of the tissue sample;   (vii) contacting the array with the section of the tissue sample;   (viii) performing RNase H-dependent PCR upon captured mRNAs of the section of the tissue sample, thereby generating a PCR-amplified DNA population comprising TCR-alpha and TCR-beta sequences; and   (ix) obtaining sequence from the PCR-amplified DNA population and an associated oligonucleotide cluster identification sequence for each DNA sequenced using a sequencing process for TCR sequence-containing PCR-amplified nucleic acids having an average read length on at least one end in excess of 200 nucleotides, thereby obtaining TCR sequences that span TCR transcript variable regions for substantially all TCR sequences obtained and obtaining sequence from the PCR-amplified DNA population of oligonucleotide cluster identification sequences associated with TCR sequences,   thereby obtaining from the tissue sample spatially-resolvable TCR sequence that spans TCR transcript variable regions;   A method for obtaining from a tissue sample spatially-resolvable TCR sequence that spans TCR transcript variable regions and macromolecule abundance data comprising:   (i) obtaining a tissue sample from a subject;   (ii) preparing a section of the tissue sample and adhering said section to a solid support;   (iii) forming an array of barcoded oligonucleotide clusters and/or an array of beads attached to barcoded oligonucleotides and contacting the section adhered to the solid support with the array;   (iv) identifying oligonucleotide cluster and/or bead array identification sequences and associated two-dimensional positions on the array of the barcoded oligonucleotide clusters and/or the array of beads attached to barcoded oligonucleotides; and   (v) obtaining the sequences of a population of macromolecules bound to the array(s) for each macromolecule sequenced, wherein the population of macromolecules comprises TCR RNA sequences, wherein TCR sequences are obtained by a process comprising RNase H-dependent PCR amplification of captured TCR RNA, thereby generating a PCR-amplified cDNA population comprising TCR-alpha and TCR-beta sequences, and obtaining sequence of the PCR-amplified cDNA population and an associated oligonucleotide cluster identification sequence for each cDNA sequenced using a sequencing process for TCR sequence-containing PCR-amplified nucleic acids having an average read length on at least one end in excess of 200 nucleotides, thereby obtaining TCR sequences that span TCR transcript variable regions for substantially all TCR sequences obtained and obtaining sequence from the PCR-amplified cDNA population of oligonucleotide cluster and/or bead array identification sequences associated with TCR sequences, thereby obtaining from the tissue sample spatially-resolvable TCR sequence that spans TCR transcript variable regions and macromolecule abundance data; and   A method for obtaining from a tissue sample spatially-resolvable T cell receptor (TCR) sequence that spans TCR transcript variable regions, the method comprising:   (i) obtaining a tissue sample from a subject;   (ii) preparing a section of the tissue sample;   (iii) providing a solid support;   (iv) contacting the solid support with a capture material, thereby forming a capture material-coated solid support;   (v) contacting the capture material-coated solid support with a population of 1-100 μm diameter beads, wherein each bead has at least 1000 attached oligonucleotides and wherein at least one attached oligonucleotide of each bead each comprises: (a) a bead identification sequence that is common to all at least 1000 oligonucleotides on each bead and (b) a poly-dT tail of sufficient length to allow for capture of poly-A-tailed RNAs via hybridization, wherein the bead identification sequence that is common to all at least 1000 oligonucleotides on each bead is either a bead identification sequence that is unique to each bead within the population of 1-100 μm diameter beads or is a bead identification sequence that is a member of a population of bead identification sequences that is sufficiently degenerate to the population of 1-100 μm diameter beads that a majority of beads within the population of 1-100 μm diameter beads each possesses a unique bead identification sequence, thereby capturing a subpopulation of the population of 1-100 μm diameter beads upon the solid support;   (vi) identifying the bead identification sequence and associated two-dimensional position on the solid support of individual beads of the subpopulation of beads attached to the solid support;   (vii) contacting the subpopulation of 1-100 μm diameter beads captured upon the solid support with of the tissue sample;   (viii) performing a reverse transcription reaction upon poly-A-tailed RNAs captured by the bead subpopulation, thereby generating a cDNA population;   (ix) contacting a selection of or all of the cDNA population with biotinylated probes capable of specifically annealing to TCR-alpha or TCR-beta sequences, and enriching for biotinylated probe-TCR complexes, thereby generating a nucleic acid population enriched for TCR-alpha and TCR-beta sequences; and   (x) obtaining sequence from the nucleic acid population enriched for TCR-alpha and TCR-beta sequences using a sequencing process for TCR sequence-containing nucleic acids having an average read length on at least one end in excess of 200 nucleotides, thereby obtaining TCR sequences that span TCR transcript variable regions for substantially all TCR sequences obtained and obtaining sequence from the nucleic acid population enriched for TCR-alpha and TCR-beta sequences of bead identification sequences associated with TCR sequences,   thereby obtaining from the tissue sample spatially-resolvable T cell receptor (TCR) sequence that spans TCR transcript variable regions.